Antigenic comparisons of selected avian reoviruses by use of the plaque-reduction neutralization assay

The antigenic interrelatedness of 3 clone-purified turkey reoviruses (NG-Turkey, 82-88, and NC-TEV) to each other and to 4 clone-purified chicken reoviruses (S1133, Co8, Fahey-Crawley, and avian type 2) was determined in reciprocal cross-neutralization tests, using polyclonal antisera and the plaque-reduction technique. The morphologic features of plaques formed under agar were studied for all 7 reoviruses, and size comparisons for turkey vs chicken isolates were made. All 3 turkey reoviruses (with the exception of NG-Turkey vs Fahey-Crawley chicken reovirus) formed plaques significantly (P less than 0.05) smaller than plaques produced by their chicken counterparts. The 3 turkey reoviruses were closely related to each other and to chicken reovirus CO8. The antigenic differences between turkey reoviruses 82-88 and NC-TEV and chicken reovirus S1133 were slight (minor subtype); however, the latter and NG-Turkey were serotypically distinct. The NG-Turkey and 82-88 turkey reoviruses were more related (minor subtype) to the Fahey-Crawley and avian type 2 chicken reoviruses, than was NC-TEV turkey reovirus (major subtype).     

新型鸭呼肠孤病毒QY株S3基因克隆与序列分析

参考GenBank新型鸭呼肠孤病毒（New-type duck reovirus,NDRV）S3基因序列设计合成一对引物,对新型鸭呼肠孤病毒QY株S3基因进行RT-PCR扩增,并对PCR产物进行了克隆和测序.结果显示扩增产物为1 104 bp,与预期的目的片段大小一致.相似性分析QY株S3基因核苷酸序列与ARV代表株、MDRV代表株和DRV代表株,相似性分别59.4％ ～ 60.0％、61.1％～ 61.3％和96.7％～ 98.6％;氨基酸的相似性分别为67.6％～ 68.7％、68.1％～68.9％和95.4％～ 98.4％.表明QY株的S3基因具有不同于ARV和MDRV的特征,分离病毒QY株是不同于禽呼肠孤病毒和番鸭呼肠孤病毒的独立基因群.     

Sequence analysis of four double-stranded RNA genomic segments reveals an orthoreovirus with a unique genotype infecting psittaciformes

This paper describes the characterization of four double-stranded ribonucleic acid segments, S1, S2, S3, and S4, of a newly identified pathogenic reovirus from parrots. The four segments share a unique 5' terminus GCUUUUC. The amino-acid sequences of the conserved sigma A and sigma NS proteins show less than 60% sequence similarity, whereas those of the outer capsid proteins sigma B and sigma C have at most 47% sequence similarity to their counterparts in other bird or bat reoviruses. In a phylogenetic analysis of the amino-acid sequences, the proteins coded for by the S1 segment, P10, P17, and sigma C, group with their homologous proteins in other avian reoviruses, whereas the major capsid protein, sigma B, and the nonstructural protein, sigma NS, show more sequence similarity to their bat reoviral counterparts. The phylogenetic relationship of sigma A with the homologous avian and bat sequences is unresolved. The possibility that the parrot reovirus has evolved from an ancestral, more batlike reovirus is discussed. It is proposed to designate this unique virus as PsRV.     

Cloning of PRL and VIP cDNAs of the Java sparrow (Padda oryzivora)

Complementary DNA (cDNA) of prolactin (PRL) and vasoactive intestinal polypeptide (VIP) of the Java sparrow were cloned and sequenced. The proximal region of the PRL promoter was also identified. Java sparrow PRL was found to have 88.3, 88.3, and 89.1% sequence identity at the cDNA level to PRL of chicken, turkey, and duck, respectively. The predicted amino acid sequence had an overall similarity with a comparable region of chicken (91.4%), turkey (88.9%) and duck (92.0%) PRL. Based on the cDNA sequence and genomic structure of the chicken PRL gene, the proximal promoter was characterized. Sequence analysis of the proximal region of Java sparrow PRL promoter revealed a high degree of similarity to that of chicken, turkey and duck PRL promoters. Moreover, cDNA of prepro-VIP was also cloned and sequenced. Java sparrow prepro-VIP shows high similarity to chicken and turkey prepro-VIP. However, the region upstream of the 5' untranslated region of Java sparrow prepro-VIP did not show similarity to that of chicken. These results suggest that the mechanisms, which regulate expression of the VIP gene, may be different between precocial and altricial birds, but expression of the PRL gene may be widely conserved in avian species.     

番鸭呼肠孤病毒非结构基因的克隆和序列分析

参考GenBank禽呼肠孤病毒(Avian Reovirus,ARV)和番鸭呼肠孤病毒(Muscovy Duck Reovirus,MDRV)非结构基因(NS)序列设计合成一对引物,对番鸭呼肠孤病毒S14和C4株NS基因进行RT-PCR扩增,克隆到pMD18-T载体中,并对克隆产物进行酶切鉴定和测序;番鸭呼肠孤病毒NS基因由1 291 bp核苷酸组成,与禽呼肠孤病毒NS基因相比,在非编码区第1155位少一个碱基,本文第一次证实1291bp是番鸭呼肠孤病毒NS基因特有的长度;番鸭呼肠孤病毒S14和C4株NS基因的5'末端和3'末端分别为5‘GCTTTT和TCATC-3',是禽类呼肠孤病毒基因末端特有的碱基序列,S14和C4株NS基因的的有效阅读框(24～1127bp)编码367个氨基酸组成的蛋白,分子量约为40kDa;番鸭呼肠孤病毒S14和C4株NS蛋白等电点分别是7.3和7.0,GC含量分别为54.26%和53.71%,番鸭呼肠孤病毒S14和C4株NS基因间核苷酸同源性为99.3%,仅有4个氨基酸差异,S14和C4与法国番鸭呼肠孤病毒89026株NS基因核苷酸同源性分别为87.8%和87.9%,与鸡关节炎病毒S1133 NS基因同源性分别为79.0%和79.3%;进化树分析表明本研究中的两株番鸭呼肠孤病毒非结构基因(NS)与番鸭呼肠孤病毒的亲缘关系比禽呼肠孤病毒近的多,建议番鸭呼肠孤病毒应归属为正呼肠孤病毒属第二个亚群中不同于禽和内尔森贝海湾呼肠病毒独立基因群.     

Molecular cloning of insulin-like growth factor-I complimentary DNA and promoter region in the domestic duck (Anas platyrhynchos)*

Complementary DNA (cDNA) and the flanking region of insulin‐like growth factor‐I (IGF‐I) of domesticated duck were cloned. The nucleotide sequence analysis of the cDNA showed seven and eight bases different, respectively, from chicken and turkey IGF‐I cDNA within the coding region. The amino acid sequence of prepro IGF‐I differed by one and two amino acids from those observed in chicken and turkey, respectively. However, no amino acid substitution was observed in the mature IGF‐I region. Sequence analysis of the promoter region and exon 1 of the duck IGF‐I revealed a high degree of similarity to that of the chicken IGF‐I gene. These results suggest that the mechanisms which regulate expression of the IGF‐I gene may be widely conserved in avian species.     

Infections Investigation of ALV and REV in Guangxi Local Poultry Breeds and Sequence Analysis of gp85 Gene of ALV Isolates

In order to investigate the infection status of avian leukosis virus (ALV) and avian reticuloendotheliosis virus (REV) in the major local breeds of Qinzhou,Guangxi,totally 953 samples of egg white,cloaca swab and serum of Ma duck,Shitou goose,Tiejiao-Ma chicken,turkey and pigeon were collected from the representing flocks and detected by the commercial ELISA kits.ALV was isolated for the ALV p27 positive samples by culturing on DF-1 cells,and gp85 gene was sequenced.The results showed that the detections of ALV were negative in the samples except those of Tiejiao-Ma chicken,while REV antibody was found positive in Ma duck,Tiejiao-Ma chicken and turkey.The nucleotide sequences of gp85 gene of two isolates shared 94.5% identity with each other,and shared 86.9% to 94.9% with reference strains.The amino acid sequences of gp85 gene of two isolates shared 91.5% identity with each other,and shared 84.0% to 91.6% with reference strains.There were many variable sites in the hyper variable region hr1 and hr2,and the vr2 and vr3 variable regions were relatively conservative.Phylogenetic tree analysis showed that the two isolates shared the highest homology with SCAU11-XG strain.     

广西地方禽类品种ALV和REV感染情况的调查及ALV分离株gp85基因序列分析

为了解广西钦州地区主要地方品种商品禽类中禽白血病病毒(ALV)、禽网状内皮组织增殖症病毒(REV)的感染情况,本试验随机采集了广西钦州地区代表饲养场的麻鸭、狮头鹅、铁脚麻鸡、火鸡、鸽子共5个品种的蛋清、肛拭子及血清样品共953份,使用ELISA商品试剂盒进行检测;然后对部分ALV p27阳性的个体采集其血清样品接种DF-1细胞进行病毒分离并测定其gp85基因的序列。结果发现,除铁脚麻鸡外其他4个非鸡禽类品种的ALV检测均为阴性;除鸽子和狮头鹅外,麻鸭、铁脚麻鸡、火鸡均检测到REV抗体阳性;获得的两株铁脚麻鸡ALV分离株gp85基因之间核苷酸同源性为94.5%,与参考株之间核苷酸同源性为86.9%~94.9%;两株分离株gp85基因之间氨基酸同源性为91.5%,与参考株之间氨基酸同源性为84.0%~91.6%。高变区hr1和hr2区存在较多可变位点,可变区vr2和vr3相对较保守。系统进化树分析结果表明这两个毒株与参考株SCAU11-XG的亲缘关系最近。     

Characterization of the sigmaB-encoding genes of muscovy duck reovirus: sigmaC-sigmaB-ELISA for antibodies against duck reovirus in ducks

The sigmaB/sigmaC-encoding genes of muscovy duck reovirus (DRV) S12 strain were cloned, sequenced, and expressed in Escherichia coli. The sigmaC-encoding gene of DRV showed only 21-22% identity to that of avian reovirus (ARV) at both nucleotide and amino acid level. The sigmaB-encoding gene of DRV comprised 1163bp with one open reading frame (ORF). The ORF comprised 1104bp and encoded 367 amino acids with a predicted molecular mass of 40.44 kDa. A zinc-binding motif and a basic amino acid motif were found within the predicted amino acid sequence of sigmaB. The identities between the S12 and ARV were 59.3-64.0% and 60.9-62.5%, respectively, at the nucleotide and deduced amino acid levels. Phylogenetic analysis of the sigmaB-encoding gene sequence indicated that S12 separated as a distinct virus relative to other avian strains. The expressed sigmaB/sigmaC fusion proteins in E. coli could be detected, approximately 45 and 50kDa, respectively, by duck anti-reovirus polyclonal serum. In addition, an ELISA (sigmaB-sigmaC-ELISA) using the expressed sigmaB-sigmaC proteins as coating antigen for detection of antibodies to DRV in ducks was developed. In comparison with the virus neutralization test and agar gel immuno-diffusion test (AGID), the sigmaB-sigmaC-ELISA showed perfect specificity and sensitivity. The sigmaB-sigmaC-ELISA did not react with the antisera to other duck pathogens, implying that these two proteins were specific in recognition of DRV antibodies. Taken together, the results demonstrated that sigmaB-sigmaC-ELISA was a sensitive and accurate method for detecting antibodies to DRV.     

中国禽(番鸭)呼肠孤病毒分离株S1基因全序列分析

采用RT-PCR技术,分别以DRV-YH、DRV-YJL两株中国禽(番鸭)呼肠孤病毒RNA为模板,扩增了S1全基因的cDNA片段.将S1 cDNA克隆到T载体后进行序列测定,测序结果表明所扩增的cDNA片段长1643个核苷酸,包含了完整的S1基因的三个开放阅读框架(ORF1,ORF2和ORF3)和基因两端的非编码区.核苷酸序列比较分析结果表明:DRV-YH与ARV-S1133,176分别有6个,10个核苷酸的差异,DRV-YJL与ARV-S1133,176分别有8个,12个核苷酸的差异,DRV-YH与DRV-YJL有4个核苷酸的差异.     

Genomic characteristics of a novel reovirus from Muscovy duckling in China

A new reovirus was isolated from a sick Muscovy duckling with hemorrhagic-necrotic lesions in the liver in Zhejiang, China in 2000 and was tentatively denoted a new type of Muscovy duck reovirus (N-MDRV ZJ00M). This reovirus was propagated in a chicken fibroblast cell line (DF-1) with obvious cytopathic effects. The reovirus's genome was 23,419 bp in length with an approximately 50% G+C content and 10 dsRNA segments encoding 12 proteins. The length of the genomic segments was similar to those of avian reoviruses (ARVs), which range from 3959 nt (L1) to 1191 nt (S4) in size. All of the segments have the conserved terminal sequences 5′-GCUUUUU…UUCAUC-3′, and all of the genome segments, with the exception of S1, apparently encoded one single primary translation product. The genome analysis revealed that the S1 segment of N-MDRV is a tricistronic gene that encodes the overlapping ORFs for p10, p18, and σC. This finding is similar to that found for ARVs but distinct from that found for classical MDRV and GRV, which have a bicistronic S4 segment that encodes p10 and σC and do not encode p18. The amino acid (aa) alignments of the putative proteins encoded by the main ORF in each segment revealed a high similarity (14.1–100%) to the counterpart proteins encoded by other ARV species from the avian orthoreoviruses (e.g., ARV, classical MDRV and N-MDRV) in the Orthoreovirus genus, particularly with N-MDRV (94.6–100%). The phylogenetic analysis of the nucleotide sequences of all 10 genome segments revealed that N-MDRV ZJ00M is distinct from all other described reovirus species groups but is a separated from the ARV (including MDRV and GRV) species within orthoreovirus species group II and grouped into the classical MDRV and GRV genogroup with the N-MDRV isolates. The MDRV genogroup can be further divided into two genotype clusters. The morphological and pathological analyses and the genetic characterization of N-MDRV ZJ00M suggest that it belongs to genotype 2 (N-MDRV). In addition, the RT-PCR assays of DRV diseased duckling and gosling samples collected from different regions of China during 2000–2013 indicate that N-MDRV is currently the prevalent genotype in China.     

新型鸭呼肠孤病毒广东分离株σC蛋白基因序列分析

从广东湛江某肉鸭养殖场发生脚软、关节肿大为临床特征和脾脏肿大、肝脏有坏死灶为病变的樱桃谷鸭病料中分离到一株新型鸭呼肠孤病毒,命名为GD693。对新型鸭呼肠孤病毒GD693株σC蛋白基因进行 RT-PCR 扩增、克隆和测序,并与参考毒株σC蛋白基因序列进行比对分析。结果显示：GD693株与新型呼肠孤病毒（NDRV）代表毒株处于进化树的同一大分支,但却处于一个单独的分支,同属于基因2型,具有相近的遗传演化关系;与NDRV代表毒株091株、TH11 株的σC蛋白基因序列进行比较分析,存在核苷酸和氨基酸的序列改变,毒株有可能发生变异或毒力增强。     

鸭源H5N1亚型高致病性禽流感病毒的分离鉴定及其HA和NA基因的生物信息分析

分离到1株 H5N1亚型高致病性禽流感病毒, 经序列测定发现HA蛋白裂解位点上插入多个连续的碱性氨基酸（PQREIRRKKR*G）,从分子上证实是一株高致病性禽流感病毒。核酸序列比较分析结果表明,分离的流感病毒HA基因与A/duck/VietNam/Ncvd1/2002(H5N1)同源率最高,达到98.8%;NA基因与A/duck/VietNam/Ncvd1/2002(H5N1) 和A/chicken/Jiangsu/cz1/2002(H5N1)同源率最高,达到98.7%。氨基酸水平上,HA与A/duck/Viet Nam/Ncvd1/2002(H5N1)同源率最高,可达99.3%;NA与A/chicken/Jiangsu/cz1/2002(H5N1)同源率最高,达98.7%。HA与NA基因的潜在糖基化位点与作者所选参比毒株一致。通过遗传进化树分析结果表明,A/duck/VietNam/Ncvd1/2002(H5N1)可能是该毒株的来源株。     

Sequence and phylogenetic analysis of M-class genome segments of novel duck reovirus NP03

We report the sequence and phylogenetic analysis of the entire M1, M2, and M3 genome segments of the novel duck reovirus (NDRV) NP03. Alignment between the newly determined nucleotide sequences as well as their deduced amino acid sequences and the published sequences of avian reovirus (ARV) was carried out with DNASTAR software. Sequence comparison showed that the M2 gene had the most variability among the M-class genes of DRV. Phylogenetic analysis of the M-class genes of ARV strains revealed different lineages and clusters within DRVs. The 5 NDRV strains used in this study fall into a well-supported lineage that includes chicken ARV strains, whereas Muscovy DRV (MDRV) strains are separate from NDRV strains and form a distinct genetic lineage in the M2 gene tree. However, the MDRV and NDRV strains are closely related and located in a common lineage in the M1 and M3 gene trees, respectively.     

Classification of Dutch and German avian reoviruses by sequencing the sigma C protein

We have amplified, cloned and sequenced (part of) the open reading frame of the S1 segment encoding the sigma C protein of avian reoviruses isolated from chickens with different disease conditions in Germany and The Netherlands during 1980 up to 2000. These avian reoviruses were analysed phylogenetically and compared with sequences of avian reoviruses in the Genbank database. The avian reoviruses could be grouped in 5 different genotyping clusters and this classification was identical when the sequences were compared of the 5' end, the 3' end or the whole open reading frame of the sigma C protein. Therefore sequencing of either part of the gene encoding the sigma C protein seems to be reliable for classification. We were unable to identify a correlation between sigma C sequences of the avian reoviruses and the disease condition they were isolated from. The sequences found in The Netherlands and in Germany are, like those in Taiwan, more dispersed than the known avian reovirus sigma C sequences in the USA and Australia. We did not establish temporal or geographic differences in the avian reoviruses studied.     

Yeast-derived sigma C protein-induced immunity against avian reovirus

Avian reoviruses (ARVs) can result in disease and economic losses in the poultry industry. Vaccines against ARV may not provide full protection and can cause adverse reactions. The coding sequence of the sigma C protein from strain S1133 of avian reovirus was expressed in Schizasaccharomyces pombe. Sigma C protein expression was demonstrated by Western blotting, and the protein was evaluated for its ability to protect specific-pathogen-free (SPF) chickens against challenge with the virulent S1133 strain. Serologic and challenge-infection data showed the efficacy of the recombinant vaccine administered orally each week for 3 consecutive wk. Sigma C protein induced antibody, as determined by enzyme-linked immunosorbent assay. Percentage (%) protection induced by the low dose (125 microg purified yeast-expressed sigma C protein/chicken) or the high dose (250 microg purified yeast-expressed sigma C protein/chicken) was 64 and 91, respectively. The commercial vaccine administered once or twice provided 82% protection. Results supported the feasibility of a plant-derived vaccine for use in poultry immunization schemes.     

中国禽(番鸭)呼肠孤病毒YB株S4基因序列分析

应用非免疫番鸭胚增殖中国禽(番鸭)呼肠孤病毒YB分离株,用LSTRIAZOL提取病毒RNA,反转录-聚合酶链反应(RT-PCR)扩增中国禽(番鸭)呼肠孤病毒YB分离株S4基因节段cDNA.将S4cDNA克隆到PMDl8-T载体上,并进行了鉴定和核苷酸序列测定.序列分析结果,克隆的S4cDNA共1 124bp,包括非编码区和完整的阅读框架.分子进化系统分析表明该毒株与禽呼肠孤病毒(鸡呼肠孤病毒)的亲缘关系较远,DRV-YB与DRV-89330同缘率为93.3%.     

北京鸭Myogenin基因部分序列的克隆及表达时间分析

利用4对引物分别对42、14日龄北京鸭胸肌组织RNA及出生后0日龄北京鸭腿肌组织和孵化22、15日龄胚胎腿肌RNA进行Myogenin基因的PCR反转录扩增，均没扩增出目的基因。资料分析表明在胚胎发育期内Myogenin基因可能只在成肌细胞分化的特定时间内表达，Myogenin基因也可在动物失去神经后或在体外培养的肌卫星细胞内表达。利用DNA扩增出了北京鸭Myogenin基因外显子1260bp部分序列，共编码86个氨基酸，编码氨基酸序列含有bHLH结构域，该结构与鸡、火鸡的同源性非常高。研究结果将为北京鸭Myogenin的表达、全序列的克隆及其分子标记的研究提供理论基础。